Fraxin in Combination with Dexamethasone Attenuates LPS-Induced Liver and Heart Injury and Their Anticytokine Activity in Mice

Background Cytokine storm syndrome (CSS) is a major cause of morbidity and mortality in people suffering from hyperinflammatory status, which diverse etiological factors, including pathogens, therapeutic interventions, malignancies, and autoimmune disorders, can instigate. Since there is limited research on the antioxidant properties of fraxin and no studies have investigated its potential as an anticytokine storm agent, it is important to note that most studies have primarily focused on proinflammatory cytokines such as IL-1β, IL-6, and TNFα during cytokine storm. However, little research discusses the role of chemokines, particularly IL-8, during cytokine storms. Therefore, further investigation is warranted into the role of fraxin as an anticytokine storm agent and the involvement of IL-8 in cytokine storms. The present study examines the preventive efficacy of fraxin and the combination of fraxin and dexamethasone (FD) in mitigating lipopolysaccharide-induced systemic inflammation in mice caused by Escherichia coli, 055: B5. Methods Five groups of ten mice were randomly assigned: LPS only group (5 mg/kg, intraperitoneally i.p.), control (normal saline N.S. 1 ml/kg, i.p.), concentrations were selected based on previous literature, fraxin (120 mg/kg, i.p.), dexamethasone (5 mg/kg, i.p.), fraxin + dexamethasone (FD) (60 mg/kg + 2.5 mg/kg, i.p.), administered one hour before LPS injection (5 mg/kg,i.p.), animals were euthanized next day, and interleukin-8 (IL-8) was quantified in serum using an enzyme-linked immunosorbent assay. The liver and heart tissues underwent histopathological analysis to assess morphological changes. For data analysis using ANOVA and Tukey post hoc tests, the Kruskal–Wallis and Mann–Whitney U tests were employed to analyze the histological results. Results A significant decline in IL-8 levels was recorded in the treatment groups almost to the same degree (p < 0.001), and the percentage of inhibition of IL-8 for fraxin, dexamethasone, and FD was 93%.92.4%, and 93%, respectively, compared to the LPS-only group. Histopathological scores were significantly reduced in liver and heart tissue (P < 0.05). Conclusions All interventions used in this study significantly reduced interleukin-8 (IL-8) levels and reduced LPS-induced liver and cardiac damage. The combination (FD) did not result in an evident superiority of either agent. More research is required to identify the possible usefulness of these agents in treating hyperinflammatory diseases, such as cytokine storms, in future clinical practice.


Introduction
Cytokine storm syndrome (CSS) is a state of excessive release of proinfammatory cytokines in which there is excessive infammation, requiring the administration of anticytokine and antiinfammatory drugs.Te immunopathology of COVID-19 is necessary to be understood, and in most critical cases, a huge increment of proinfammatory cytokines has been recorded, causing CSS and activating acute respiratory distress (ARD) toward multiorgan destruction [1].
Infammation is tissues' natural biological response to chemical and mechanical stimuli or infectious agents, most often instigated by a diverse range of bacterial species.Pathogenic conditions such as chronic infections and the infammatory response can cause notable detriments to the host, namely, lipopolysaccharide (LPS), a fundamental constituent of the external membrane of Gram-negative bacteria that has been widely used to develop an infammatory model [2].
Following the introduction of LPS in experimental animals, several endotoxic efects, such as fever, leukopenia, leukocytosis, thrombocytopenia, disseminated intravascular coagulation, and hemodynamic alterations, may terminate in fatal shock [3].
Te initial step in the classical interactions between lipopolysaccharide (LPS) and host cells involves binding LPS to the lipopolysaccharide binding protein (LBP).Subsequently, this complex forms a bond with a receptor complex comprising CD14, MD2, and toll-like receptor 4 (TLR4), thereby initiating a signaling cascade that elicits the secretion of proinfammatory cytokines [4].
However, previous studies have confrmed that macrophages and endothelial cells can also internalize lipopolysaccharide (LPS) via routes not dependent on toll-like receptor 4 (TLR4).After being internalized, lipopolysaccharide (LPS) can form a bond with the cytosolic receptor caspase-4/5 in humans and the corresponding caspase-11 in mice.Caspases-4/5 form oligomers and initiate the assembly of the infammasome-3, which consists of the nucleotidebinding domain, leucine-rich family, and pyrin domaincontaining protein [5].
Tis assembly then leads to the activation of infammatory caspase-1, leading to the subsequent release of interleukin-1β.Caspases-4/5 are known to initiate the activation of perforin gasdermin D and purinergic receptor P2X7, hence leading to cell lysis and pyroptosis induction.Pyroptosis is a prominent contributor to the infammatory response and impairment of the pulmonary endothelial barrier in cases of sepsis [6].Neutrophil granulocytes represent the predominant cellular constituents of the innate immune system.Te adequate activation of these cellular entities is fundamental to efectively eliminating pathogens.Upon exposure to a potent chemoattractant, neutrophils undergo activation and subsequently migrate to the site of infammation, thus constituting the primary cellular defense mechanism against such insults [7].Interleukins, such as IL-8, are frequently used as a diagnostic and prognostic indicator for conditions related to infection (specifcally septic conditions) and other types of infammation (such as traumatic conditions).Given the potential importance of IL-8-mediated neutrophil activation and its associated mechanisms in systemic infammation, there is signifcant scientifc and clinical interest in understanding these processes.Specifcally, such insights may provide valuable information on possible pharmacological targets to regulate an excessive infammatory response resulting from IL-8 actions [8].In recent times, there have been numerous revelations about natural products found within plants that exhibit unique structural attributes and demonstrate diverse biological activities.Te use of these therapeutic agents has furnished numerous valuable primary compounds for the development of new drugs.Polyphenols, particularly favonoids, have been found to manifest physiological and pharmacological efects comprising antioxidant, antiinfammatory, antidiabetic, antibacterial, antitumor, and neuroprotective properties [9].Te feld of drug discovery is based on phytochemicals as the primary focus for developing new small-molecule chemical entities.Plant-derived natural products are recognized as a signifcant and crucial class of compounds that contribute to developing established and emerging pharmacological treatments.Contemporary reports indicate that many pharmaceutical companies have reduced their emphasis on exploring potential therapeutic agents derived from natural sources [10].Fraxin (isolated from Cortex Fraxini) is a 7, 8-dihydroxy-6-methoxy coumarin, and 8-D glucopyranoside is characterized by a wide range of activities, including antioxidant, analgesic, antimicrobial, antiviral, and immunomodulatory [11].Fraxin has been found to exhibit signifcant pharmacological versatility, showing dual protection against oxidative stress and infammation in various variables [12].As such, it can be hypothesized that fraxin may facilitate a fundamental role in attenuating interleukin-8 release and conferring protection against LPS-induced harm in the murine population [13].Te current research aims to evaluate Fraxin's hepatoprotective and cardioprotective properties and combine it with dexamethasone in mice exposed to lipopolysaccharide (LPS).Te study investigated the infuence of the said substances on interleukin-8 levels.

Chemicals and Reagents. Te chemicals and reagents
utilized in this study are listed in Table 1.

Animals. Swiss
Albino BALB/c mice (ffty males, 20-25 g, 7-8 weeks) were used (animals purchased from the Drug Quality Control and Research National Center, Animal House Facility, Iraq), randomly housed fve in each cage with a 12-hour light/dark cycle, and mice were acclimated for a week and kept in a pathogen-free place with a temperature of 23 °C ± 2 °C and a humid atmosphere.Enough food and water were available all the time for all the rodents.Te Animal Ethics Committee of Al-Nahrain University, College of Pharmacy, accepted the experimental protocol under issue number Nah.Co. Pha.12.

Study Design.
Following a period of adaptation lasting one week, a total of 50 male mice were divided into fve groups at random allocation, each consisting of 10 mice, as shown in Table 2; all interventions were administered one hour before induction intraperitoneally (ip) with LPS (5 mg/ kg).[14].

Serum Collection and Quantifcation of Cytokine Levels.
After 24 hours, after lipopolysaccharide administration (LPS), mice were subjected to anesthesia using a high dose of inhalational diethyl ether.Blood samples were then collected from the jugular veins and centrifuged at a speed of 2857 × g

Measurement of IL-8 Cytokine
Level.Using a Shanghai YL Biont ELISA kit, the IL-8 level was quantifed in the serum of treated mice.Fraxin and fraxin + dexamethasone (FD) activity was assessed in LPS-induced mice pretreated one hour before LPS induction.Figure 1 shows that IL-8 levels were signifcantly reduced in treatment groups almost to the same degree (p < 0.001), and the percentage of inhibition of interleukin-8 for fraxin, dexamethasone, and FD was 93%, 92.4%, and 93%, respectively, compared to the LPS-only control.

Histopathological Analysis.
For all treatment groups, histopathological fndings for the liver are shown in Figure 2 and for heart in Figure 3, and the control group that received NS only is shown in Figure 2

Discussion
In the current study, we tested the activity of fraxin, dexamethasone, and their fraxin + dexamethasone (FD) combination on a model of systemic infammation developed by injecting LPS intraperitoneally (5 mg/kg) once.Te observed inhibitory percentages for the interleukin-8 were 93% for fraxin, 92.4% for dexamethasone, and 93% for FD, compared to the control group that was exposed solely to LPS.All  treatments dramatically reduced IL-8 levels in mouse serum; this efect could be attributed to the broad pharmacological action of fraxin, which included antiinfammatory, antioxidant, immune-modulatory, antiaging, anticancer, and antibacterial properties [12].Te present study builds on a previous research that demonstrated the ability of fraxin to act as an antagonist to LPS-triggered cytokine release.Li and his colleagues conducted two separate studies that established potential mechanisms through which fraxin may facilitate this biological response.Specifcally, their fndings suggested that fraxin may achieve its efects by manipulating signaling pathways associated with infammatory cells, such as the NF-κB pathway and NLRP3.Furthermore, it was found that the concentrations of tumor necrosis factor-α and interleukin-6 in serum and selected anatomical structures, specifcally the lung and liver, were decreased [17].
In histopathological aspects, the results of this investigation indicate that the administration of fraxin, dexamethasone, and their combination successfully mitigated the deleterious efects of lipopolysaccharide (LPS) in various organs.From the previous literature, the administration of fraxin has been shown to mitigate histopathological damage to the liver resulting from cisplatin, evidenced by a reduction in necrosis of coagulation, hydropic degeneration, sinusoidal dilatation, and hyperemia.[18].Also, fraxin exhibited a notable protective efect in countering liver damage induced by CCl 4 , resulting in a dose-dependent reduction in pathological scores [11].
Te ethyl acetate fraction of Fraxinus xanthoxyloides, denoted as FXE, demonstrated a certain degree of cardioprotective efects at a low dose of 150 mg/kg compared to CCl 4 .On the contrary, the high dosage of FXE at 300 mg/kg and silymarin exhibited a protective efcacy comparable to that of the control after CCl 4 administration.At both doses administered, FXE did not induce any histological alterations in cardiac tissues, indicating that it does not have a discernible impact on normal tissue morphology [19].
Te administration of corticosteroids is associated with specifc considerations, including decreased immune response and increased susceptibility to infections.In contrast, the use of corticosteroids for recovery from cytokine storms related to viral infection has shown promise, and it is important to consider that their immunosuppressive efects could potentially exacerbate viral load and increase susceptibility to secondary infections [20].From the current study, we can recommend that fraxin alone and in combination with a low dose of dexamethasone may be a safer option since it will not afect the normal function of the immune system.

Conclusion and Recommendations
Te fndings of our investigation revealed that fraxin, dexamethasone, and the combination FD exhibit considerable efcacy in reducing levels of interleukin-8 (IL-8) in vivo in mice subjected to lipopolysaccharide (LPS) challenge through modulation of various signaling pathways.Signifcantly, from the previous literature, these compounds inhibit TLR-mediated NF-κB signaling.Histopathological analysis showed that all interventions employed in this investigation reduced LPS-induced severe liver and cardiac damage.Histopathological scores exhibited a marked reduction compared to the group receiving only LPS treatment.Concurrent use of fraxin and dexamethasone in LPS-induced mice did not result in discernible superiority of either agent individually, as evidenced by equivalent quantifcation of IL-8 and fndings of the comparable histopathological analysis in both the liver and the heart.Still, future studies could consider fraxin an anticytokine storm agent.Additional research on fraxin and its combinations with other phytochemicals is essential to evaluate the efect of these agents on antiinfammatory cytokines such as IL-10; diferent concentrations and diferent combinations can be considered in future work and research to fnd the precise way these drugs work and determine the potential applicability of these agents in the management of hyperinfammatory conditions, such as cytokine storm, in clinical practice.
Te present study encountered certain limitations that must be acknowledged.Each treatment and the combination 6 Advances in Virology were evaluated using a single concentration.It should be noted that the limited sample size of animal groups and the specifc location of blood withdrawal may signifcantly impact cytokine measurement levels.Additionally, the sampling timingcan increase the chance of measurement variability.
(a); the liver section shows a normal appearance of central vein and sinusoids.Figure 3(a) shows the normal cardiac muscle fber of the heart

Figure 1 :Figure 2 :
Figure 1: Te level of IL-8 in LPS-induced mice serum after 24 hours.Treatment with fraxin, dexamethasone, and fraxin + dexamethasone (FD); data are presented as mean ± SEM; * highly signifcant P < 0.001 in comparison to LPS alone, and # highly signifcant P < 0.001 in comparison to control, n � 10.Te red line indicates groups injected with LPS (5 mg/kg), 1 hour after treatment.

Figure 4 :Figure 5 :
Figure 4: Te histopathological scoring for all liver tissue treatment groups, with data presented as mean rank ± SEM. * Treatment groups demonstrated statistical signifcance at p < 0.05 compared to the lipopolysaccharide LPS group and # signifcant at (p < 0.05) for LPS compared to the control, n � 5.

Table 1 :
Te chemicals and reagents used in this study.

Table 2 :
An overview of the treatments employed, including details on dosage and route of administration.